Bottom Line:
Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface.These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit.Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi.

Affiliation: Department of Physiology and Pharmacology, University of Toledo College of Medicine, Health Science Campus, Toledo, OH 43614, USA.

ABSTRACTHere, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

fig6: Expression of wild-type rat α1 and D371N, but not mCBM, in TCN23-19 cells restored Cav1 distribution. Cells were cotransfected with caveolin-CFP and YFP-tagged wild-type rat α1 (α1 wt), D371N, or mCBM. Images were taken by confocal microscope, and pseudo-color was assigned to the images to show colocalization. Red arrowhead: rescued cells; blue arrowhead: cells that were not rescued by either rat α1 or its mutants. A set of representative images of three separate experiments is shown. Bar, 5 μm.

Mentions:
To seek further evidence to support the above conclusion, we repeated the transient transfection and imaging analyses using different α1 constructs. As shown in Fig. 6 A, in TCN23-19 cells rescued by wild-type α1 (pointed by red arrowhead), the expressed Cav1-CFP was targeted to the plasma membrane. On the other hand, in the same image we found that Cav1-CFP–positive vesicles were largely retained in the cytoplasm in a cell that did not express a significant amount of YFP-α1 (pointed by blue arrowhead). Moreover, expression of the YFP-mCBM α1 mutant failed to restore the cellular distribution of Cav1-CFP (Fig. 6 C), which is consistent with the fractionation studies presented in Fig. 5.

fig6: Expression of wild-type rat α1 and D371N, but not mCBM, in TCN23-19 cells restored Cav1 distribution. Cells were cotransfected with caveolin-CFP and YFP-tagged wild-type rat α1 (α1 wt), D371N, or mCBM. Images were taken by confocal microscope, and pseudo-color was assigned to the images to show colocalization. Red arrowhead: rescued cells; blue arrowhead: cells that were not rescued by either rat α1 or its mutants. A set of representative images of three separate experiments is shown. Bar, 5 μm.

Mentions:
To seek further evidence to support the above conclusion, we repeated the transient transfection and imaging analyses using different α1 constructs. As shown in Fig. 6 A, in TCN23-19 cells rescued by wild-type α1 (pointed by red arrowhead), the expressed Cav1-CFP was targeted to the plasma membrane. On the other hand, in the same image we found that Cav1-CFP–positive vesicles were largely retained in the cytoplasm in a cell that did not express a significant amount of YFP-α1 (pointed by blue arrowhead). Moreover, expression of the YFP-mCBM α1 mutant failed to restore the cellular distribution of Cav1-CFP (Fig. 6 C), which is consistent with the fractionation studies presented in Fig. 5.

Bottom Line:
Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface.These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit.Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi.

Affiliation:
Department of Physiology and Pharmacology, University of Toledo College of Medicine, Health Science Campus, Toledo, OH 43614, USA.

ABSTRACTHere, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.